The removal of non-coding sequences from RNA (splicing) is required for the expression of most genes in higher eukaryotes as well as some genes in bacteria. Control of the rate of RNA splicing is believed to determine steady-state levels of mRNA in yeast and in some pathogenic viruses such as influenza virus and AIDS virus. We will study the splicing of group I introns (intervening RNA sequences) in a model system--yeast mitochondria. Group I introns are similar in structure to the RNA genomes of infectious agents such as viroids and hepatitis delta virus. The splicing of this class of introns can occur without proteins in vitro. In the cell, however, proteins are known to be involved in some cases. We will study the role of proteins in intron splicing. Specifically we will: (1) Characterize the autocatalytic splicing of certain group I introns. (2) Generate deletions in introns to determine which sequences are required for splicing. (3) Fractionate trans-acting splicing factors (proteins or RNA- protein particles) and determine their effects on RNA splicing in vitro. (4) Use immunologic, electrophoretic and chromatographic techniques to characterize proteins or complexes which stimulate splicing. Because of its utility for biochemical and genetic manipulation, we will employ baker's yeast (Saccharomyces cerevisiae) for these studies. The introns we will investigate occur in the cob gene, which encodes apocytochrome b, and the oxi3 gene, which encodes subunit 1 of cytochrome c oxidase. Both are part of the mitochondrial genome. This research will help explain how RNA processing modulates the levels of energy-harnessing enzymes of the inner mitochondrial membrane. Mitochondria provide most of the chemical energy in animal cells. Consequently, this project examines the long-term regulation of energy production, a process governed at the level of gene expression.